Aim 1. We demonstrated previously that sleep can vary among flies having identical genotypes subjected to identical environmental conditions. We quantified this variability as the sensitivity to the environment. Sensitivity to the environment is strongly correlated with sleep duration; flies having short night sleep were far more sensitive to random environmental perturbations than longer-sleeping flies. As this sensitivity is heritable, we identified many polymorphisms putatively involved in its regulation. How these polymorphisms cause variable sleep phenotypes among flies having the same genotype and subjected to the same environmental conditions is not known, but one distinct possibility is that environmental sensitivity might alter gene expression. Therefore, our first goal is to determine whether transcripts are altered among flies with identical genotypes subjected to a common environment. If significant changes in gene expression among identical individuals exist, then it would indicate a possible mechanism by which different sleep phenotypes might arise in individuals of the same genotype. We examined the changes in endogenous gene expression in a sample of 16 lines randomly chosen from the Drosophila Genetic Reference Panel (DGRP). Each line was subjected to the standard environment of sucrose-cornmeal food, 25 deg. C, 60% humidity, and 12:12 hour light:dark cycle. Three biological replicates of the same environment were made. Individual virgin males and females were harvested, and total RNA was extracted. We constructed RNA sequence libraries and successfully sequenced RNA from 750 of the 768 individual flies that were frozen; 726 samples passed genotype and sex quality control guidelines. As there are no clear-cut strategies for the normalization and analysis of RNA-Seq data, we are in the process of comparing seven different popular normalization methods for raw count data and six separate data filtering and analysis approaches.